HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY OF OLIGONUCLEOTIDES
HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY OF OLIGONUCLEOTIDES
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DOI：
10.1016/0003-2697(85)90324-0
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发表时间：
1985-01-01
影响因子：
2.9
通讯作者：
REGNIER, FE
中科院分区：
文献类型：
--
作者：
DRAGER, RR;REGNIER, FE
几种类型的高性能二氧化硅基支持物被发现可有效分离多核苷酸。这些材料的主要区别在于键合相的类型及其附着到二氧化硅载体上的方法。一种方法是通过硅氧烷键将固定相基团偶联至表面。该技术简单，可产生高容量和分辨率的材料，但其键合相稳定性较差。另一种方法是吸附交联成表面膜的低分子量聚乙烯亚胺 (PEI)。固定相通过薄膜在多个位点的吸附而固定在适当的位置。之前关于该材料的报告显示了包含多达 30 个碱基的寡核苷酸的分辨率。本文报道了在制备寡核苷酸和 tRNA 物质的 HP-IEC 色谱柱时进一步优化 PEI 键合化学。研究发现离子交换基质的季铵化可将寡核苷酸的分辨率从 30 个碱基提高到 50 个碱基。发现相同的支持物能够解析多种 tRNA。使用 60 至 120 分钟的硫酸铵梯度，在小型（0.42 × 5 cm）柱上实现分离。初始溶剂为 15% 乙腈的 0.05 M 磷酸钾溶液 (pH 5.9)。向初始溶剂中添加 1 M 硫酸铵用于制备最终溶剂。
Several types of high-performance silica-based supports were found to be effective in the separation of polynucleotides. The principal difference in these materials is the type of bonded phase and the method by which it is attached to the silica support. One approach is the coupling of stationary-phase groups to the surface through siloxane bonding. This technique is simple and produces a material of high capacity and resolution, but it suffers from poor bonded-phase stability. An alternative approach is the adsorption of low-MW polyethylene imines (PEI) that are crosslinked into a surface film. The stationary phase is held in place by adsorption of the film at multiple sites. A previous report on this material showed the resolution of oligonucleotides containing up to 30 bases. This paper reports further optimization of the PEI bonding chemistry in the preparation of HP-IEC columns for oligonucleotides and tRNA species. Quaternization of the ion-exchange matrix was found to increase resolution of oligonucleotides from 30 to 50 bases. The same support was found to be capable of resolving multiple tRNA species. Separations were achieved on small (0.42 .times. 5 cm) columns, using a 60- to 120-min ammonium sulfate gradient. The initial solvent was 15% acetonitrile in 0.05 M potassium phosphate (pH 5.9). The addition of 1 M ammonium sulfate to the initial solvent was used to prepare the final solvent.